Senescence was determined either by flow cytometry or immunostaining as two independent methods. For the flow cytometric analysis, cells were incubated for 30 min in Bafilomycin A1 and afterwards C12FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37 °C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α–tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56 °C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc., Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
Chicken anti rabbit alexa 594
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Chicken anti-rabbit Alexa-594 is a secondary antibody conjugated with the Alexa Fluor 594 dye. It is used to detect and visualize rabbit primary antibodies in a variety of applications, such as immunofluorescence, Western blotting, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
C12fdg, by Thermo Fisher Scientific (4 mentions)
α tubulin, by Abcam (4 mentions)
Anti h3k9me3, by Merck Group (4 mentions)
Anti hmga2 and p21, by Cell Signaling Technology (4 mentions)
Alexa 488 chicken anti mouse, by Thermo Fisher Scientific (4 mentions)
Vectashield, by Vector Laboratories (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (4 mentions)
Celltrace far red, by Thermo Fisher Scientific (3 mentions)
Celltrace oregon green, by Thermo Fisher Scientific (3 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (3 mentions)
Bafilomycin a1, by Thermo Fisher Scientific (2 mentions)
Ba lomycin a1, by Thermo Fisher Scientific (2 mentions)
Axio plan 2 uorescence microscope, by Zeiss (2 mentions)
Goat anti chicken alexa 488, by Thermo Fisher Scientific (2 mentions)
Rabbit anti brn3, by Santa Cruz Biotechnology (2 mentions)
Goat anti chicken alexa 594, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse alexa 594, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Chicken anti β gal, by Abcam (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
9 protocols using chicken anti rabbit alexa 594
1
Senescence and Phagocytosis Analysis
2
Senescence Detection by Flow Cytometry and Immunostaining
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Senescence was determined either by ow cytometry or immunostaining as two independent methods.
For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
3
Senescence and Phagocytosis Characterization
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Senescence was determined either by flow cytometry or immunostaining as two independent methods. For the flow cytometric analysis, cells were incubated for 30 minutes in Bafilomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany).
Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 fluorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
4
Senescence Analysis by Flow Cytometry and Immunostaining
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Senescence was determined either by ow cytometry or immunostaining as two independent methods. For the ow cytometric analysis, cells were incubated for 30 minutes in Ba lomycin A1 and afterwards C 12 FDG (Invitrogen, Auckland, New Zealand) was added for 60 min at 37°C. For senescence analysis by immunostaining anti-H3K9me3 (Millipore, Darmstadt, Germany), anti-HMGA2 and p21 (Cell Signaling, Leiden, Netherlands) antibodies were used. Cytoskeleton was stained by α-tubulin (Abcam, Cambridge, UK). Secondary antibodies were Alexa 488 chicken anti mouse or Alexa 594 chicken anti rabbit (Molecular Probes, Darmstadt, Germany). Phagocytosis experiments were performed using CellTrace Oregon Green (CTOG) (Invitrogen, Auckland New Zealand) for the living cells and CellTrace Far Red (CTFR) (Invitrogen, Auckland, New Zealand) for heat (56°C) for camptothecin treated cells. Cell nuclei were stained by DAPI (Roche, Grenzach-Wyhlen, Germany) and slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, USA). Cell images were acquired by a Zeiss Axio Plan 2 uorescence microscope (Zeiss, Göttingen, Germany). Nuclear size was analyzed with Biomas software (MSAB, Erlangen, Germany).
5
Immunofluorescence Staining of Cryosectioned Eyes
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Eyes were harvested and fixed in 4% PFA for 2 hours prior to cryoprotection in 25% sucrose-PBS overnight. Eyes were embedded in OCT, and cryosectioned at 12 μm and mounted on SuperFrost Plus slides prior to immunofluorescence. Standard immunofluorescence procedures were followed.9 (link) Primary antibodies include: chicken anti-β-gal (Abcam Cambridge, MA; ab9361), rabbit anti-β-tubulin III (Covance, Princeton, NJ; PRB-435P), rabbit anti-Brn3 (Santa Cruz Biotechnology, Santa Cruz, CA; sc28595), and rabbit anti-Sox2 (Abcam; ab97959). Secondary antibodies used were (all from Invitrogen): chicken anti-rabbit Alexa-594, goat anti-chicken Alexa-488, goat anti-chicken Alexa-594, goat anti-mouse Alexa-488, goat anti-mouse Alexa-594, goat anti-rabbit Alexa-488, and goat anti-rabbit Alexa-594. Nuclei were stained using 1 μg/ml of Hoechst 33342 (Sigma, St Louis, MO; 14533). Slides were mounted with Prolong Gold (Invitrogen) antifade reagent and imaged using an Olympus BX61 microscope and the InVivo software (Olympus), or alternatively using confocal laser scanning microscopy with a Leica SP5 II microscope (Leica Microsystems) and LAF software (Leica Microsystems).
6
Immunohistochemistry of Retinal Tissues
7
Multicolor Immunofluorescence Labeling
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Rabbit-anti-mouse-Alexa594 (Invitrogen #A11062); goat-anti-mouse-Alexa488 (Invitrogen #A11001); rabbit-anti-mouse-Alexa633 (Invitrogen #A21063); chicken-anti-rabbit-Alexa594 (Invitrogen #A21442); goat-anti-rabbit-Alexa488 (Invitrogen #A11008); goat-anti-rabbit-Alexa633 (Invitrogen #A21070).
8
Multimarker Immunofluorescence Staining Protocol
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Primary antibodies used for immunostaining included those for EGFP (Santa Cruz Biotechnology), MMP7 (R & D Systems, Minneapolis, MN, USA), cytokeratin (Leica, Solms, Germany), CD34 (Abcam, Cambridge, MA, USA), GRP78 (Enzo Life Sciences, Lausen, Switzerland), cleaved caspase-3, cleaved caspase-4, cleaved caspase-9, cleaved caspase-12, Cox IV (all from Cell Signaling Technology, Danvers, MA, USA); secondary antibodies included goat anti-mouse HRP (Santa Cruz Biotechnology), goat anti-rabbit HRP (Santa Cruz Biotechnology), donkey anti-goat HRP (Santa Cruz Biotechnology), chicken anti-rat AP (Santa Cruz Biotechnology), chicken anti-rabbit AP (Santa Cruz Biotechnology), chicken anti-mouse AP (Santa Cruz Biotechnology), goat anti-rabbit Alexa 488/594 (Invitrogen), rabbit anti-goat Alexa 594 (Invitrogen), chicken anti-rabbit Alexa 594 (Invitrogen) and chicken anti-mouse Alexa 594 (Invitrogen). Immunostaining was performed using these antibodies. The sections were counter-stained with hematoxylin or nuclear fast red (Vector, Burlingame, CA, USA). For double staining, EGFP, MMP7, cytokeratin, CD34 and PAS staining was performed following TUNEL staining or cleaved caspase-3 staining.
9
Immunofluorescence Labeling of P2X2 and P2X3
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Coverslips with plated neurons were fixed with 4% PFA and blocked with superblock (Thermo Fisher Scientific) for 30 min. Human HNSCC tissues were resected and fixed with 4% PFA, dehydrated, embedded in paraffin, and cut into 8-μm sections. Sections were then deparaffinized and blocked with superblock. H&E staining was performed to confirm cancer lesions. For immunofluorescence labeling, neurons or tissue sections were then incubated for 24 h at 4°C in rabbit anti-P2X3 (1:500, Alomone Labs) and goat anti-P2X2 (1:500, Santa Cruz Biotechnology). The sections were then washed in phosphate-buffered saline (PBS) with Triton X-100 and incubated in secondary antibody chicken anti-rabbit Alexa-594 (1:1000, Invitrogen) and donkey anti-goat Alexa-488 (1:1000, Invitrogen) in a dark chamber for 2 hours at room temperature. Control experiments were performed by incubation in secondary antibody alone and by applying P2X2 blocking peptides (Santa Cruz Biotechnology), and P2X3 blocking peptides (Alomone Labs). The coverslips or sections were washed and visualized with images acquired using a Nikon Ti Eclipse microscope (Nikon).
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